INHIBITORY EFFECT OF ETHANOL ON HEPATOCYTE GROWTH FACTOR-INDUCED DNA-SYNTHESIS AND CA2+ MOBILIZATION IN RAT HEPATOCYTES

Hepatocyte growth factor (HGF) is the most potent mitogen identified f or hepatocytes and is thought to be an important growth factor in the regulation of liver regeneration. Its effects are mediated through a t yrosine kinase receptor, the product of c-met proto-oncogene. One of t he downstream signaling processes activated by HGF is phospholipase C- gamma. HGF stimulation of liver cells causes formation of inositol 1,4 ,5-triphosphate, which releases Ca2+ from intracellular Ca2+ ([Ca2+](i )) stores, and causes elevation of cytosolic Ca2+ levels. It is known that liver regeneration is inhibited by both acute and chronic ethanol (EtOH) treatment. We investigated the effect of EtOH on HGF-induced D NA synthesis and mobilization of [Ca2+](i) in rat hepatocytes in prima ry culture. DNA synthesis was monitored by [H-3]thymidine incorporatio n in primary cultures of hepatocytes 42 hr after stimulation with HGF. HGF concentration required for maximum DNA synthesis was 0.3 to 1 ng/ ml, and DNA synthesis was inhibited by 100 mM EtOH at HGF concentratio ns in the range of 0.1 to 5 ng/ml. This inhibition was strongest (45 t o 47% inhibition) at a low concentration of HGF (0.1 to 0.3 ng/ml) and decreased at an HGF concentration >1 ng/ml. HGF-induced changes in [C a2+](i) were measured in single fura 2-loaded hepatocytes by fluoresce nce imaging techniques. The Ca2+ response induced by HGF (0.3 to 5 ng/ ml) was inhibited by EtOH, with an EC(50) of similar to 50 mM. Analysi s of Ca2+ response patterns in individual cells indicated that EtOH su ppressed the number of responsive cells and made Ca2+ responses more t ransient, but did not affect peak [Ca2+](i) elevation; thus suggesting an inhibition at the level of phospholipase C-gamma-activation. These data indicate that inhibition by EtOH of the response of liver cells to HGF may contribute to the inhibitory effect of EtOH on liver regene ration.